2QVO.xds
XDS data reduction
dataset 1
Using "generate_XDS.INP ../../APS/22-ID/2qvo/ACA10_AF1382_1.0???" we obtain:
JOB= XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT ORGX= 1996.00 ORGY= 2028.00 ! check these values with adxv ! DETECTOR_DISTANCE= 125.000 OSCILLATION_RANGE= 1.000 X-RAY_WAVELENGTH= 1.90000 NAME_TEMPLATE_OF_DATA_FRAMES=../../APS/22-ID/2qvo/ACA10_AF1382_1.0??? ! REFERENCE_DATA_SET=xxx/XDS_ASCII.HKL ! e.g. to ensure consistent indexing DATA_RANGE=1 360 SPOT_RANGE=1 180 ! BACKGROUND_RANGE=1 10 ! rather use defaults (first 5 degree of rotation) SPACE_GROUP_NUMBER=0 ! 0 if unknown UNIT_CELL_CONSTANTS= 70 80 90 90 90 90 ! put correct values if known INCLUDE_RESOLUTION_RANGE=50 0 ! after CORRECT, insert high resol limit; re-run CORRECT FRIEDEL'S_LAW=FALSE ! This acts only on the CORRECT step ! If the anom signal turns out to be, or is known to be, very low or absent, ! use FRIEDEL'S_LAW=TRUE instead (or comment out the line); re-run CORRECT ! remove the "!" in the following line: ! STRICT_ABSORPTION_CORRECTION=TRUE ! if the anomalous signal is strong: in that case, in CORRECT.LP the three ! "CHI^2-VALUE OF FIT OF CORRECTION FACTORS" values are significantly> 1, e.g. 1.5 ! ! exclude (mask) untrusted areas of detector, e.g. beamstop shadow : ! UNTRUSTED_RECTANGLE= 1800 1950 2100 2150 ! x-min x-max y-min y-max ! repeat ! UNTRUSTED_ELLIPSE= 2034 2070 1850 2240 ! x-min x-max y-min y-max ! if needed ! ! parameters with changes wrt default values: TRUSTED_REGION=0.00 1.2 ! partially use corners of detectors; 1.41421=full use VALUE_RANGE_FOR_TRUSTED_DETECTOR_PIXELS=7000. 30000. ! often 8000 is ok MINIMUM_ZETA=0.05 ! integrate close to the Lorentz zone; 0.15 is default STRONG_PIXEL=6 ! COLSPOT: only use strong reflections (default is 3) MINIMUM_NUMBER_OF_PIXELS_IN_A_SPOT=3 ! default of 6 is sometimes too high REFINE(INTEGRATE)=CELL BEAM ORIENTATION ! AXIS DISTANCE ! parameters specifically for this detector and beamline: DETECTOR= CCDCHESS MINIMUM_VALID_PIXEL_VALUE= 1 OVERLOAD= 65500 NX= 4096 NY= 4096 QX= .0732420000 QY= .0732420000 ! to make CORRECT happy if frames are unavailable DIRECTION_OF_DETECTOR_X-AXIS=1 0 0 DIRECTION_OF_DETECTOR_Y-AXIS=0 1 0 INCIDENT_BEAM_DIRECTION=0 0 1 ROTATION_AXIS=1 0 0 ! at e.g. SERCAT ID-22 this needs to be -1 0 0 FRACTION_OF_POLARIZATION=0.98 ! better value is provided by beamline staff! POLARIZATION_PLANE_NORMAL=0 1 0
Now we run "xds_par". This runs to completion. We should at least inspect, using XDS-Viewer, the file FRAME.cbf since this shows us the last frame of the dataset, with boxes superimposed which correspond to the expected locations of reflections.
The automatic spacegroup determination (CORRECT.LP) comes up with
LATTICE- BRAVAIS- QUALITY UNIT CELL CONSTANTS (ANGSTROEM & DEGREES) REINDEXING TRANSFORMATION CHARACTER LATTICE OF FIT a b c alpha beta gamma * 44 aP 0.0 41.2 53.5 53.5 90.3 90.1 90.1 -1 0 0 0 0 1 0 0 0 0 -1 0 * 31 aP 0.8 41.2 53.5 53.5 89.7 90.1 89.9 1 0 0 0 0 1 0 0 0 0 1 0 * 25 mC 1.4 75.4 75.8 41.2 90.0 90.1 90.0 0 1 -1 0 0 -1 -1 0 -1 0 0 0 * 35 mP 1.8 53.5 41.2 53.5 90.1 90.3 90.1 0 -1 0 0 1 0 0 0 0 0 1 0 * 23 oC 3.1 75.4 75.8 41.2 90.0 90.1 90.0 0 1 -1 0 0 -1 -1 0 -1 0 0 0 * 20 mC 3.9 75.8 75.4 41.2 90.1 90.0 90.0 0 1 1 0 0 1 -1 0 -1 0 0 0 * 34 mP 5.1 41.2 53.5 53.5 90.3 90.1 90.1 1 0 0 0 0 0 1 0 0 -1 0 0 * 33 mP 5.3 41.2 53.5 53.5 90.3 90.1 90.1 -1 0 0 0 0 1 0 0 0 0 -1 0 * 32 oP 6.1 41.2 53.5 53.5 90.3 90.1 90.1 -1 0 0 0 0 1 0 0 0 0 -1 0 * 21 tP 7.3 53.5 53.5 41.2 90.1 90.1 90.3 0 1 0 0 0 0 -1 0 -1 0 0 0 39 mC 249.8 114.5 41.2 53.5 90.1 90.3 69.0 1 -2 0 0 1 0 0 0 0 0 1 0
indicating at most tetragonal symmetry, shortly after this calculates R-factors for these lattices:
SPACE-GROUP UNIT CELL CONSTANTS UNIQUE Rmeas COMPARED LATTICE-
NUMBER a b c alpha beta gamma CHARACTER
5 75.8 75.4 41.2 90.0 90.0 90.0 900 40.8 5882 20 mC
* 75 53.5 53.5 41.2 90.0 90.0 90.0 469 8.4 6313 21 tP
89 53.5 53.5 41.2 90.0 90.0 90.0 282 39.2 6500 21 tP
21 75.4 75.8 41.2 90.0 90.0 90.0 506 39.8 6276 23 oC
5 75.4 75.8 41.2 90.0 90.1 90.0 901 40.7 5881 25 mC
1 41.2 53.5 53.5 89.7 90.1 89.9 1699 8.2 5083 31 aP
16 41.2 53.5 53.5 90.0 90.0 90.0 521 39.8 6261 32 oP
3 53.5 41.2 53.5 90.0 90.3 90.0 931 8.2 5851 35 mP
3 41.2 53.5 53.5 90.0 90.1 90.0 918 40.7 5864 33 mP
3 41.2 53.5 53.5 90.0 90.1 90.0 918 40.9 5864 34 mP
1 41.2 53.5 53.5 90.3 90.1 90.1 1699 8.2 5083 44 aP
thus suggesting spacegroup #75 but we should know that this does not take screw axes into account. Therefore we use "pointless xdsin XDS_ASCII.HKL" and are told that this is actually spacegroup P4_2 (# 77). Alternatively, we could have inspected the list further down in CORRECT.LP:
REFLECTIONS OF TYPE H,0,0 0,K,0 0,0,L OR EXPECTED TO BE ABSENT (*) -------------------------------------------------------------------- H K L RESOLUTION INTENSITY SIGMA INTENSITY/SIGMA #OBSERVED 0 0 1 41.248 0.8487E+01 0.1339E+01 6.34 4 0 0 3 13.749 -0.7977E-03 0.3786E+01 0.00 4 0 0 4 10.312 0.1305E+06 0.4660E+04 27.99 1 0 0 5 8.250 0.1318E+01 0.6316E+01 0.21 4 0 0 6 6.875 0.2939E+05 0.5284E+03 55.61 4 0 0 7 5.893 0.5439E+01 0.9235E+01 0.59 4 0 0 8 5.156 0.1298E+05 0.2371E+03 54.73 4 0 0 9 4.583 0.3308E+02 0.1327E+02 2.49 4 0 0 10 4.125 0.3809E+05 0.6867E+03 55.47 4 0 0 11 3.750 -0.1987E+02 0.1767E+02 -1.12 4 0 0 12 3.437 0.5539E+04 0.1097E+03 50.48 4 0 0 13 3.173 0.2144E+01 0.2071E+02 0.10 4 0 0 14 2.946 0.2717E+04 0.6252E+02 43.46 4 0 0 15 2.750 0.1350E+02 0.2482E+02 0.54 4 0 0 16 2.578 0.1178E+04 0.4383E+02 26.88 4 0 0 17 2.426 -0.7420E+01 0.3549E+02 -0.21 4 0 0 18 2.292 0.4104E+03 0.4681E+02 8.77 4
and realize that this also tells us that the spacegroup is 77, not 75.
After his comes the table that tells us the quality of our data:
NOTE: Friedel pairs are treated as different reflections.
SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas Rmrgd-F Anomal SigAno Nano
LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr
6.06 4189 556 560 99.3% 2.4% 2.7% 4187 66.74 2.6% 1.1% 74% 1.841 247
4.31 7575 1008 1008 100.0% 2.6% 2.9% 7575 62.90 2.8% 1.2% 62% 1.463 473
3.53 9468 1283 1283 100.0% 3.4% 3.2% 9468 53.37 3.6% 1.7% 41% 1.200 612
3.06 11364 1540 1540 100.0% 5.1% 4.7% 11364 34.45 5.5% 3.1% 17% 0.995 739
2.74 12628 1695 1695 100.0% 10.2% 10.4% 12628 17.09 11.0% 7.9% 2% 0.797 819
2.50 14121 1916 1916 100.0% 21.5% 23.1% 14121 8.42 23.1% 17.1% -4% 0.691 926
2.31 15155 2079 2079 100.0% 46.6% 50.5% 15155 3.92 50.2% 38.6% -1% 0.734 1010
2.16 12185 2104 2228 94.4% 113.3% 117.0% 12178 1.44 124.7% 119.0% 5% 0.753 1018
2.04 5134 1601 2347 68.2% 274.7% 291.2% 4913 0.40 325.5% 400.7% 1% 0.608 606
total 91819 13782 14656 94.0% 5.7% 5.9% 91589 20.24 6.2% 15.0% 12% 0.897 6450
NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES 93217
NUMBER OF REJECTED MISFITS 1391
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS 0
NUMBER OF ACCEPTED OBSERVATIONS 91826
NUMBER OF UNIQUE ACCEPTED REFLECTIONS 13784
So the anomalous signal goes to about 3.3 A (which is where 30% would be, in the "Anomal Corr" column), and the useful resolution goes to 2.16 A, I'd say (pls note that this table treats Friedels separately; merging them increases I/sigma by another factor of 1.41).
For the sake of comparability, from now on we use the same axes (53.03 53.03 40.97) as the deposited PDB id 2QVO.
We could now modify XDS.INP to have
JOB=CORRECT ! not XYCORR INIT COLSPOT IDXREF DEFPIX INTEGRATE CORRECT SPACE_GROUP_NUMBER= 77 UNIT_CELL_CONSTANTS= 53.03 53.03 40.97 90.000 90.000 90.000
and run xds again, to obtain the final CORRECT.LP and XDS_ASCII.HKL with the correct spacegroup, but the statistics in 75 and 77 are the same, for all practical purposes (the 8 reflections known to be extinct do not make much difference).
Following this, we create XDSCONV.INP with the lines
SPACE_GROUP_NUMBER= 77 ! can leave out if CORRECT already ran in #77 UNIT_CELL_CONSTANTS= 53.03 53.03 40.97 90 90 90 ! same here INPUT_FILE=XDS_ASCII.HKL OUTPUT_FILE=temp.hkl CCP4
and run "xdsconv", and then
f2mtz HKLOUT temp.mtz<F2MTZ.INP cad HKLIN1 temp.mtz HKLOUT output_file_name.mtz<<EOF LABIN FILE 1 ALL END EOF
which gives us output_file_name.mtz, which we rename to xds-2ovo-1-F.mtz. Similarly, using
OUTPUT_FILE=temp.hkl CCP4_I
we end up with a MTZ file with intensities, which we rename to xds-2ovo-1-I.mtz.
dataset 2
This works exactly the same way as dataset 1. The table in CORRECT.LP is
NOTE: Friedel pairs are treated as different reflections.
SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas Rmrgd-F Anomal SigAno Nano
LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr
6.06 3925 547 560 97.7% 3.0% 3.3% 3922 56.13 3.3% 1.4% 80% 1.874 242
4.31 7498 1000 1000 100.0% 2.8% 3.4% 7498 56.91 3.0% 1.2% 65% 1.473 469
3.53 9407 1291 1291 100.0% 3.4% 3.5% 9407 52.39 3.7% 1.6% 55% 1.276 616
3.06 11005 1526 1526 100.0% 4.1% 3.9% 11005 42.13 4.4% 2.2% 39% 1.211 732
2.74 12569 1701 1701 100.0% 5.7% 5.7% 12569 28.38 6.1% 3.7% 4% 0.881 822
2.50 14020 1904 1904 100.0% 9.0% 9.9% 14020 17.92 9.7% 6.3% 3% 0.741 921
2.31 15101 2081 2081 100.0% 17.0% 19.0% 15101 9.83 18.3% 12.7% -5% 0.682 1011
2.16 11693 2080 2202 94.5% 39.4% 40.8% 11682 4.00 43.6% 45.8% 10% 0.791 1003
2.04 5152 1607 2345 68.5% 85.6% 93.5% 4943 1.21 101.3% 129.6% 10% 0.718 615
total 90370 13737 14610 94.0% 4.2% 4.5% 90147 24.22 4.6% 7.3% 22% 0.956 6431
NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES 92690
NUMBER OF REJECTED MISFITS 2318
NUMBER OF SYSTEMATIC ABSENT REFLECTIONS 0
NUMBER OF ACCEPTED OBSERVATIONS 90372
NUMBER OF UNIQUE ACCEPTED REFLECTIONS 13738
Dataset 2 is definitively better than dataset 1.
SHELXC/D/E structure solution
This is done in a subdirectory of the XDS data reduction directory (either dataset "1" or "2", and we can also try it in a xscale subdirectory). Here, we generate XDSCONV.INP (I used MERGE=TRUE, sometimes the results are better that way) and run xdsconv and SHELXC.
#!/bin/csh -f cat > XDSCONV.INP <<end INPUT_FILE=../XDS_ASCII.HKL OUTPUT_FILE=temp.hkl SHELX MERGE=TRUE FRIEDEL'S_LAW=FALSE end xdsconv shelxc j <<end SAD temp.hkl CELL 53.03 53.03 40.97 90 90 90 SPAG P42 MAXM 2 end
This writes j.hkl, j_fa.hkl and j_fa.ins. However, we overwrite j_fa.ins now (these lines are just the ones that hkl2map would write):
cat > j_fa.ins <<end TITL j_fa.ins SAD in P42 CELL 0.98000 53.03 53.03 40.97 90.00 90.00 90.00 LATT -1 SYMM -Y, X, 1/2+Z SYMM -X, -Y, Z SYMM Y, -X, 1/2+Z SFAC S UNIT 128 SHEL 999 3.0 FIND 3 NTRY 100 MIND -1.0 2.2 ESEL 1.3 TEST 0 99 SEED 1 PATS HKLF 3 END end
and then
shelxd j_fa
This gives best CC All/Weak of 37.28 / 21.38 for dataset 1, and best CC All/Weak of 37.89 / 23.80 for dataset 2, and .
Next we run G. Sheldrick's beta-Version of SHELXE Version 2011/1:
shelxe.beta j j_fa -a -q -h -s0.55 -m20 -b
and the the inverse hand:
shelxe.beta j j_fa -a -q -h -s0.55 -m20 -b -i
One of these (and it's impossible to predict which one!) solves the structure, the other gives bad statistics.
Some important lines in the output: for dataset 1, I get
78 residues left after pruning, divided into chains as follows: A: 78 CC for partial structure against native data = 36.54 % ... Estimated mean FOM and mapCC as a function of resolution d inf - 4.49 - 3.55 - 3.10 - 2.81 - 2.61 - 2.45 - 2.32 - 2.22 - 2.13 - 2.03 <FOM> 0.763 0.784 0.743 0.682 0.632 0.620 0.621 0.600 0.519 0.416 <mapCC> 0.890 0.936 0.916 0.893 0.838 0.827 0.847 0.858 0.836 0.768 N 721 728 722 720 719 738 749 721 674 721 Estimated mean FOM = 0.639 Pseudo-free CC = 65.26 % Density (in map sigma units) at input heavy atom sites Site x y z occ*Z density 1 0.0293 0.3394 0.3145 16.0000 19.09 2 -0.1598 0.3789 0.3723 12.7456 15.78 3 -0.1413 0.4707 0.3704 9.4720 7.85 4 -0.2238 0.1590 0.4520 9.2176 9.96 5 0.0387 0.4228 0.3134 1.6608 1.28 Site x y z h(sig) near old near new 1 0.0293 0.3392 0.3148 19.1 1/0.02 2/10.34 4/11.66 4/11.66 5/12.88 2 -0.1564 0.3740 0.3757 16.4 2/0.35 5/4.38 4/5.45 1/10.34 3/12.03 3 -0.2146 0.1625 0.4495 11.0 4/0.53 2/12.03 5/15.84 1/16.92 4/17.39 4 -0.1386 0.4748 0.3671 8.1 3/0.29 5/2.67 2/5.45 1/11.66 1/11.66 5 -0.1829 0.4512 0.3605 5.9 3/2.47 4/2.67 2/4.38 1/12.88 1/13.92
and for dataset 2,
80 residues left after pruning, divided into chains as follows: A: 80 ... CC for partial structure against native data = 46.31 % Estimated mean FOM and mapCC as a function of resolution d inf - 4.49 - 3.55 - 3.10 - 2.81 - 2.61 - 2.45 - 2.32 - 2.22 - 2.13 - 2.02 <FOM> 0.726 0.703 0.695 0.704 0.706 0.713 0.667 0.572 0.535 0.503 <mapCC> 0.850 0.863 0.857 0.899 0.900 0.908 0.866 0.805 0.828 0.814 N 719 721 725 719 713 736 755 722 673 705 Estimated mean FOM = 0.654 Pseudo-free CC = 67.40 % Density (in map sigma units) at input heavy atom sites Site x y z occ*Z density 1 0.1613 0.5298 0.4706 16.0000 22.30 2 0.1266 0.3414 0.5281 14.4576 17.03 3 0.3453 0.2833 0.6078 11.1760 11.69 4 0.0318 0.3665 0.5267 6.6512 8.45 5 0.0499 0.3350 0.5280 5.8208 5.38 Site x y z h(sig) near old near new 1 0.1605 0.5316 0.4699 22.4 1/0.11 2/10.61 4/11.62 4/11.62 5/12.61 2 0.1258 0.3407 0.5328 17.4 2/0.20 5/3.83 4/5.39 1/10.61 3/12.02 3 0.3367 0.2831 0.6107 13.2 3/0.47 2/12.02 5/15.41 1/17.15 4/17.33 4 0.0269 0.3630 0.5241 9.3 4/0.33 5/2.78 2/5.39 1/11.62 1/11.62 5 0.0575 0.3206 0.5182 8.2 5/0.95 4/2.78 2/3.83 1/12.61 1/14.10
clearly indicating that the structure can be solved with each of the two datasets individually.
Can we do better?
data reduction
The safest way to optimize the data reduction is to look at external quality indicators. Internal R-factors, and even the correlation coefficient of the anomalous signal are of comparatively little value. A readily available external quality indicator is CC All/CC Weak as obtained by SHELXD.
WFAC1 was already discussed above. Another candidate for optimization is MAXIMUM_ERROR_OF_SPOT_POSITION. By default this is 3.0 . In the case of these data, this default appears to be too small, because the STANDARD DEVIATION OF SPOT POSITION (PIXELS) (as reported by IDXREF, INTEGRATE and CORRECT after refinement) is quite high (1.5 and more). This prevents XDS from using all the reflections for geometry refinement. In general, it makes sense to use MAXIMUM_ERROR_OF_SPOT_POSITION= (at least 3 times the STANDARD DEVIATION OF SPOT POSITION (PIXELS))
I found that MAXIMUM_ERROR_OF_SPOT_POSITION=6.0 significantly improved the internal statistics (mostly the r-factors, but not so much the correlation coefficient of the anom signal), and improved CC All/CC Weak indicators (to more than 40). SHELXE then produces significantly better and more complete models. Try for yourself!
Optimization does improve things as much as it often does: recycling of GXPARM.XDS to use as XPARM.XDS, and thus imposing the lattice symmetry in the geometry refinement in INTEGRATE. These findings my correspond to the fact that in P1 the angles do not refine to 90.0xx or 89.9xx degrees. In other words, the metric symmetry is not as well fulfilled as it should be. This results in fairly large deviations from the ideal P42 positions; the refinement of cell parameters in P1 partly compensates for this. I have however no idea why this deviation from metric symmetry could occur.
structure solution
The resolution limit for SHELXD could be varied. For SHELXE, the solvent content could be varied, and the number of autobuilding cycles, and probably also the high resolution cutoff. Furthermore, it would be advantageous to "re-cycle" the file j.hat to j_fa.res, since the heavy-atom sites from SHELXE are more accurate than those from SHELXD, as the phases derived from the poly-Ala traces are quite good (compare the density columns of the two consecutive heavy-atom lists!).
Limits
With dataset 2, I tried to use 270 frames but could not solve the structure using the above SHELXC/D/E approach (not even with MAXIMUM_ERROR_OF_SPOT_POSITION=6.0). With 315 frames, it was possible.